The present invention relates generally to materials and methods for engineering microorganisms such as yeast and bacteria whereby single or multiple copies of endogenous or exogenous genes of interest may be added to the microorganism without adding drug resistance gene markers by using a novel suite of recombinant yeast integration plasmids. Specifically, the methods and materials of this invention may be used to produce new strains of yeast which overproduce inositol but which lack drug resistance gene markers. The resulting recombinant microorganism does not contain any drug resistance gene markers and in the case of yeast the genetically modified organism could be considered xe2x80x9cgenerally recognized as safexe2x80x9d (xe2x80x9cGRASxe2x80x9d) if only endogenous Saccharomyces cerevisiae or other GRAS organism genes are introduced.
Biosafety of genetically modified microorganisms for uses in human food, animal feed or plant fertilizers is a major issue to be considered in the development and application of emerging biotechnologies. The United States, Europe, and Japan are especially sensitive to the potential biohazards from foods and additives from biotechnological sources. Most of the current recombinant DNA processes use circular DNA molecules called plasmids as cloning vectors to introduce homologous or heterologous genetic information into microorganisms such as bacteria, yeast, plant, and animal cells. Plasmids have the advantage that they exist normally in prokaryotic and lower eukaryotic cells in single or multimeric forms, which means that a certain gene located on such a plasmid could exist in the cell in multicopy form, which may result in a higher expression of the proteins encoded by these genes. In such instances plasmids serve as vectors on which the new genetic property or properties can be engineered and then be subsequently transferred into a host cell. In order to detect whether such a transfer has been successful and also to maintain the plasmid in the transformed cell, the plasmids must also contain a so-called selection gene marker. A selection gene marker is typically a gene that confers resistance to an antibiotic or another substance that kills or prevents the growth of cells that do not contain such a plasmid. A selection marker can also confer other phenotypic traits that can be selected such as the color of colonies, a new enzymatic activity, as well as others.
Plasmid vectors can be replicated in the host cell if they contain autonomous replication sequences recognized by the host DNA synthesis machinery, or if they are able to integrate in the genome of the host cell. The first type of such plasmids, also known as episomal plasmids, can be amplified in large numbers inside a single host cell and their replication is independent of the host chromosomal replication during the cell cycle. The other type of such plasmids, also known as integrative plasmids, can only be replicated and maintained if they are integrated into the host cell genome. Many plasmid vectors used to transform eukaryotic host cells are known as shuttle episomal vectors, meaning that they contain prokaryotic and eukaryotic DNA sequences that allow their episomal amplification in either type of host cells. There are also combinations of prokaryotic-episomal/eukaryotic-integrative shuttle vectors.
The DNA amplification step of all episomal plasmid vectors required to transform eukaryotic cells is preferably carried out in bacterial hosts due to their faster growth rate and ease of purification of the plasmidic DNA. Most plasmids used in molecular biology (such as pBR and pUC-derived plasmids) contain as a selection marker the bacterial drug resistance marker for the ampr or bla gene (See, Sutcliffe, J. G., et al., Proc. Natl. Acad. Sci. U.S.A. 75:3737 (1978), the disclosure of which is incorporated herein by reference). The bla gene encodes an enzyme named TEM-1, which is a widespread plasmidic xcex2-lactamase for narrow-spectrum cephalosporins, cefamandole, and cefoperazone and all the anti-gram-negative-bacterium penicillins except temocillin.
Unfortunately, when using the most common recombinant DNA plasmid vectors to transform microorganisms these drug resistance gene markers are inherently transferred along with the gene or genes that are of interest to the host microorganism. Consequently there is potential for the transfer of bacterial drug resistance genes from a genetically modified organism (xe2x80x9cGMOxe2x80x9d) used as a feed additive to a wild or pathogenic bacteria that is present in the normal intestinal flora of the animal being fed, thereby transforming the normal microbial flora of the animal into new and potentially dangerous antibiotic resistant strains. For example, the TEM-1 enzyme was first reported in 1965 from an E. coli isolate and is now the most common xcex2-lactamase found in enterobacteria. Resistance in more than 50% of Ampr E. coli clinical isolates is due to TEM-1.
The world is currently facing similar problems due to indiscriminant overuse or misuse of antibiotics to treat common human and animal illnesses. This overuse/misuse has created drug resistance in bacterial strains that are no longer responsive to available antibiotics. See, Levy, S. B., Scientific American, March 1998, pp. 46-53, the disclosure of which is incorporated herein by reference. The FDA as well as the World Health Organization (WHO) and European regulatory agencies are concerned about biotechnology-derived products that could lead to the uncontrolled dissemination of drug resistance genes into the environment, hence posing a potential health problem. Therefore, genetically engineered organisms, for example, recombinant yeast containing bacterial drug resistance gene markers are considered potentially biohazardous and could not be used as food or as a feed additive without carrying out a comprehensive health and environmental risk assessment for its use.
Another disadvantage of plasmids that contain bacterial antibiotic selection gene markers for use in human or animal food fermentation is that antibiotics are costly and they must be present in the medium in which the bacterial host has to be cultivated. In response to this problem xe2x80x9cfood-gradexe2x80x9d vectors have been developed. See, for example, U.S. Pat. No. 5,627,072, the disclosure of which is incorporated herein by reference.
In the specific case of yeast, and in particular those of the genus Saccharomvces, a recombinant molecule containing a drug resistance gene marker would not be considered xe2x80x9cgenerally recognized as safexe2x80x9d (xe2x80x9cGRASxe2x80x9d) by the Food and Drug Administration and would be considered to be a potential biohazard unless a risk assessment is performed on the recombinant yeast. The GRAS classification is important when choosing a microorganism to industrially manufacture a product such as inositol that is also GRAS (see, 21 C.F.R. 184.1370(c)), since a byproduct of the fermentation is the yeast biomass itself or its extract. Therefore, if the yeast biomass is biohazard-free, it can itself be sold for profit as a valuable feed or food additive, which is the case for the genus Saccharomyces. Conversely, a non-GRAS biomass, such as certain yeasts of the genus Candida, for example, Candida boidinii, can be considered biohazardous waste, and therefore such biomass has to be disposed of by costly procedures which add to the production cost and which could potentially impact the environment. Currently, biohazardous biomass has to be either incinerated or disposed of according to the NIH guidelines for the treatment of biohazardous waste. Therefore, having a xe2x80x9cGRASxe2x80x9d regulatory status is an important aspect to be considered for a successful industrial biotechnology process.
Fermentation technology can produce many compounds at a lower manufacturing cost than the present chemical technologies, with the added advantage of being environmentally safe. Such an example is the production of inositol and inositol-containing metabolites. Industrial methods to produce inositol have been based on chemical conversion of phytic acid (inositol hexaphosphoric acid ester) to myo-inositol and phosphate using corn mill factory steep water. See, Bartow, E., et al., Ind. Eng. Chem. 30:300 (1938) the disclosure of which is incorporated herein by reference. The first practical industrial methods, still in use in Asia today, involve the breakdown of phytic acid with high heat, high pressures, and very acidic conditions. See, U.S. Pat. No. 2,112,553, the disclosure of which is incorporated herein by reference. Such processes produce waste products that impact the environment thereby adding to production costs due to the necessity, for treatment procedures of such waste byproducts.
Inositol, or more specifically, the most abundant isomer, myo-inositol, is presently used in non-milk based infant formula, premixed vitamin supplements for humans, and is used as feed additive for animals and aquaculture applications. Recently, however, new uses for inositol have been suggested for alternative treatments of human diseases such as diabetes type II, folate-resistant neural tube defects (NTDs), manic depressive disorders, and obsessive compulsive disorders (xe2x80x9cOCD""sxe2x80x9d).
Inositol and inositol derivatives can be produced by fermentation of genetically modified yeast. White, M. J., et al., J. Biol. Chem. 2:863 (1991), (hereinafter referred to as xe2x80x9cWhite, et al. (1991)xe2x80x9d), and U.S. Pat. Nos. 5,529,912, and 5,599,701, the disclosures of which are incorporated herein by reference, describe genetically modified yeast of the genus Saccharomyces which has a functional stable recombinant DNA sequence that prevents the expression of the OPI1 gene, a negative regulator of phospholipid biosynthesis which results in the overexpression and excretion of inositol. There is described therein a complete deletion of the open reading frame (xe2x80x9cORFxe2x80x9d) region of the OPI1 gene, which results in the constitutively depression of the gene INO1, the result of which is the overproduction and excretion of inositol and overproduction of inositol-containing metabolites and/or phospholipids. Many of the other coregulated enzymes involved in phospholipid biosynthesis are also expressed at high levels. Using these methods, a diploid Opixe2x88x92 strain with its two endogenous copies of the INO1 gene was constructed accordingly (See, U.S. Pat. No. 5,599,701), designated as the YS2 strain, and was deposited at the American Type Culture Collection located at 10801 University Boulevard, Manassas, Va., under accession number 74033. U.S. Pat. No. 5,529,912 further describes an Opixe2x88x92 diploid S. cerevisiae strain designated as YS3 (ATCC accession number 74034) containing multiple (3-6) copies of the INO1 gene. U.S. Pat. No. 5,296,364, the disclosure of which is incorporated herein by reference, describes a method for increasing production of inositol into the growth media using the S. cerevisiae strains designated as YS2 and YS3.
U.S. Pat. No. 5,618,708, the disclosure of which is incorporated herein by reference, describes mutants of the yeast genus Candida which excrete inositol into the growth media. Some members of the genus Candida, however, are not considered GRAS, as stated above.
However, even though wild type S. cerevisiae is considered a GRAS organism, new genetically modified strains such as the YS3 strain described in White, et al. (1991) and U.S. Pat. Nos. 5,529,912 and 5,599,701 is not considered to be GRAS because it contains bacterial drug resistance gene markers that were inserted in its genome during the insertion of recombinant plasmids carrying extra copies of the INO1 gene.
In order to solve the above-described limitations of the present genetic engineering technology, new recombinant DNA techniques and materials have been developed that allow for genetic metabolic engineering of yeast without adding any heterologous drug resistance genes in the DNA recombination process. They also allow for the stable integration of genes of interest into yeast, such as even more copies of the INO1 gene than has been previously possible, and the integrations are done at different loci in the genome hence increasing the genetic stability of the resulting strain. These novel recombinant strains could be considered GRAS and contain improved nutritional qualities.
Accordingly, it is a primary object of the present invention to eliminate the use of drug resistance gene markers in plasmids for genetically engineering yeast destined for human food or animal feed supplements.
Another object of the present invention is to construct recombinant microorganisms so that genes of interest are added to the host microorganism without adding any drug resistance genes or other undesirable genes as selection gene markers.
Another object of the present invention is to use Saccharomyces cerevisiae genes as selection gene markers for plasmid amplification in bacterial hosts, such as LEU2, which are considered GRAS.
Yet another object of the present invention is to produce a yeast of the genus Saccharomyces containing one or multiple copies of a specific endogenous yeast gene targeted to one or more different loci in the yeast genome but lacking drug resistance gene markers.
Still another object of the present invention is to produce yeast containing a group of genes that codes for enzymes that make up a step of or an entire metabolic pathway that the host yeast does not contain without adding any drug resistance gene markers.
Another object of the present invention is to provide a suite of yeast integration plasmids which can be stably integrated into a yeast genome without carrying over any drug resistance or other undesirable exogenus genes.
Another object of the present invention is to provide a suite of vectors which use non-lethal genes such as nutritional yeast genes as selectable markers for DNA amplification in bacteria or yeast and non-lethal genes such as yeast genes as targeting gene markers for integration into auxotrophic yeast strains.
Another object of the present invention is to provide a recombinant yeast cell of the genus Saccharomyces that is free of drug resistance genes that overproduces inositol and inositol metabolites at levels above those of any other yeast strains.
Still yet another object of the present invention is to provide yeast that is enriched as to inositol, inositol-containing metabolites, phosphorylated and/or methylated forms of inositol or any of its stereoisomers, or phosphatidyl-inositols that does not contain any drug resistance genes.
Another object of the present invention is to produce prototrophic recombinant microorganisms that are free of drug resistance genes and which can be grown in minimal media without the addition of antibiotics or nutritional supplements such as amino acids.
These and other objects of the present invention are achieved by one or more of the following embodiments.
In one aspect, the invention features a method of stably inserting at least one gene of interest into a specific locus in the genome of a host haploid yeast cell without adding any drug resistance genes to the host to form a genetically modified diploid, comprising:
constructing host haploid yeast strains of opposite mating types to contain a non-lethal, selectable gene mutation, a target gene mutation, in at least one gene of either of the haploids;
constructing at least one or a suite of a plurality of yeast integration plasmids, wherein each member plasmid comprises a selection gene marker which allows for replication of the plasmid in a plasmid amplification host and for selection of the transformed colonies of the host; a targeting gene marker for directing the insertion of the plasmid into a specific mutated homologous locus, a target gene mutation, in the host yeast genome; a gene of interest that is desired to be expressed in the host yeast; and an origin of DNA replication functional in a plasmid amplification host, such that the yeast integration plasmids do not contain any drug resistance genes;
amplifying each of the yeast integration plasmids in the plasmid amplification host wherein the plasmid amplification host has a selectable phenotype which is released by the selection gene marker of the yeast integration plasmid upon its transformation into the plasmid amplification host;
purifying the amplified yeast integration plasmids;
transforming opposite mating types of the host yeast haploids with yeast integration plasmids such that a targeting gene marker of a yeast integration plasmid directs the integration of the integration plasmid to the target gene mutation in the host to which it is homologous; and
mating the transformed haploid yeast cells of opposite mating types to form a diploid cell.
In another aspect, the invention features a yeast integration plasmid, comprising:
a selection gene marker;
a targeting gene marker;
a gene of interest; and
an origin of DNA replication, wherein the plasmid does not contain any drug resistance genes.
In another aspect, the invention features a yeast cell of the genus Saccharomyces having an opixe2x88x92 phenotype genetically engineered to contain one or more target gene mutations comprising ade2, his3, leu2, lys1, met15, trp1, and ura3 which cause auxotrophic phenotypes in the host which can be rescued by insertion of one or more yeast integration plasmids which carry a targeting gene marker which comprises the corresponding functional gene which is homologous to the target gene mutation in the host and which rescues the corresponding auxotrophies of the host.
In a preferred embodiment, the yeast integration plasmids carry the INO1 gene.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiment, and from the claims.